Use of signal transduction inhibitors and combination therapies for the prevention or treatment of cancer and angiogenesis related diseases

ABSTRACT

The invention provides improved compositions (e.g., combinations of signal transduction inhibitors) and methods for the prevention, stabilization, or treatment of cancer or other angiogenesis related diseases. In particular, the present methods use combination therapies to modulate the expression or activity of multiple mRNA molecules or proteins associated with angiogenesis or cancer in a mammal (e.g., a human)

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was funded by grant PO1-CA-80124-02 from the NationalInstitutes of Health. The government may have certain rights in theinvention.

BACKGROUND OF THE INVENTION

In general, the invention features methods for the selection of apreferred therapy (e.g., one or more signal transduction inhibitors) forthe treatment of a particular cancer patient or group of cancerpatients. The invention also provides improved methods for the treatmentand prevention of a variety of cancers and angiogenesis related diseasesin mammals (e.g., humans).

Cancer is one of the leading causes of death. Some cancers respondpoorly to chemotherapy or have initial favorable responses tochemotherapy but later develop resistance after repeated chemotherapytreatments. For example, some tumors survive anti-angiogenic therapytargeted against a single angiogenic factor, such as vascularendothelial growth factor (VEGF), by switching their dependence to otherfactors. In addition, multidrug-resistance genes found in some cancercells enable the cells to pump out drugs, rendering the cancersresistant to multiple classes of drugs. Many of the current treatmentsthat destroy cancerous cells also affect normal cells, resulting in avariety of possible side-effects, such as nausea, vomiting, low bloodcell counts, increased risk of infection, hair loss, and ulcers inmucous membranes. In addition to cancer, there are a variety ofangiogenesis related diseases that are associated with excessive orinsufficient vascular growth.

Thus, improved therapies are needed that result in a higher incidence ofremissions and longer lengths of remissions. Other desirable therapiesprevent the initial occurrence of a cancer or another angiogenesisrelated disease or prevent the recurrence of a cancer or angiogenesisrelated disease. Preferably, the therapies produce few adverseside-effects and are useful for the treatment of a variety of cancers orangiogenesis related diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide improved methods fortreating and preventing cancer and angiogenesis related diseases. Inparticular, these methods involve the selection of a preferred signaltransduction inhibitor or a preferred combination therapy (e.g., acombination of signal transduction inhibitors) for a patient diagnosedwith, or at increased risk for, a cancer or an angiogenesis relateddisease based on the patient's expression profile of cancer orangiogenesis related genes, such as pro-angiogenic or anti-angiogenicgenes. Preferably, the therapy modulates the expression of multiplegenes (e.g., 5, 10, 15, or more cancer or angiogenesis related genes) inthe subject in an amount sufficient to prevent, stabilize, or treatcancer or an angiogenesis related disease.

Accordingly, in a first aspect, the invention provides a method ofselecting a combination therapy for the treatment, stabilization, orprevention of a cancer or an angiogenesis related disease in a mammal.This method involves analyzing the expression profile of more than onemRNA and/or protein in a sample obtained from the mammal. A therapy isselected that includes two or more compounds (e.g., signal transductioninhibitors) that each (i) decrease the expression level or activity ofan mRNA or protein that has a higher than normal expression level in themammal and/or (ii) increase the expression level or activity of an mRNAor protein that has a lower than normal expression level in the mammal.

In a related aspect, the invention provides a method of selecting asignal transduction inhibitor for the treatment, stabilization, orprevention of a cancer or an angiogenesis related disease in a mammal.This method involves analyzing the expression profile of one or moremRNA molecules and/or proteins in a sample obtained from the mammal. Asignal transduction inhibitor is selected that (i) decreases theexpression level or activity of an mRNA or protein that has a higherthan normal expression level in the mammal and/or (ii) increases theexpression level or activity of an mRNA or protein that has a lower thannormal expression level in the mammal.

In another related aspect, the invention provides a method forpreventing, delaying, or treating a cancer or an angiogenesis relateddisease in a mammal. This method involves analyzing the expressionprofile of more than one mRNA and/or protein in a sample obtained fromthe mammal. A therapy is selected that includes two or more compounds(e.g., signal transduction inhibitors) that each (i) decrease theexpression level or activity of an mRNA or protein that has a higherthan normal expression level in the mammal and/or (ii) increase theexpression level or activity of an mRNA or protein that has a lower thannormal expression level in the mammal. The selected therapy isadministered to the mammal in an amount sufficient to treat, stabilize,or prevent the cancer or angiogenesis related disease.

In yet another related aspect, the invention provides another method forpreventing, delaying, or treating a cancer or an angiogenesis relateddisease in a mammal. This method involves analyzing the expressionprofile of one or more mRNA molecules and/or proteins in a sampleobtained from the mammal. A signal transduction inhibitor is selectedthat (i) decreases the expression level or activity of an mRNA orprotein that has a higher than normal expression level in the mammaland/or (ii) increases the expression level or activity of an mRNA orprotein that has a lower than normal expression level in the mammal. Theselected signal transduction inhibitor is administered to the mammal inan amount sufficient to treat, stabilize, or prevent the cancer orangiogenesis related disease.

The invention also provides methods for classifying subjects involved ina clinical trial for a combination chemotherapeutic or angiogenesismodulating therapy (e.g., a combination of signal transductioninhibitors) based on the subjects' expression profiles. This methodallows the subjects who are most likely to benefit from a particularcombination therapy to be included in the corresponding subgroup for theclinical trial. Thus, this method enables the association of aparticular expression profile with improved drug efficacy to bedemonstrated or confirmed in humans.

According to this aspect of the invention, a method is provided forstratification of subjects involved in a clinical trial of a combinationtherapy that includes two or more compounds (e.g., signal transductioninhibitors) for the treatment, stabilization, or prevention of a canceror an angiogenesis related disease in a mammal. This method involvesanalyzing the expression profile of a sample obtained from a subject anddetermining the presence of a lower or higher than normal expressionlevel for more than one mRNA and/or protein in the sample before,during, or after the clinical trial. The presence of a particularexpression profile in the subject places the subject into a subgroup forthe clinical trial or excludes the subject from a subgroup for theclinical trial.

In a related aspect, the invention provides a method for stratificationof subjects involved in a clinical trial of a signal transductioninhibitor for the treatment, stabilization, or prevention of a cancer oran angiogenesis related disease in a mammal. This method involvesanalyzing the expression profile of a sample obtained from a subject anddetermining the presence of a lower or higher than normal expressionlevel for one or more mRNA molecules and/or proteins in the samplebefore, during, or after the clinical trial. The presence of aparticular expression profile in the subject places the subject into asubgroup for the clinical trial or excludes the subject from a subgroupfor the clinical trial.

The invention also features combination therapies that are useful forthe prevention, stabilization, or treatment of cancer or angiogenesisrelated diseases. Because these therapies contain multiplepharmaceutically active compounds, the therapies modulate the expressionor activity of numerous cancer or angiogenesis related molecules.

In one such aspect, the invention features a pharmaceutical compositionthat includes at least 2, 3, 5, 7, 10, 15, or more pharmaceuticallyactive compounds (e.g., signal transduction inhibitors) that eachmodulate the expression or activity of at least 1, 3, 5, 10, 15, 20, 30or more cancer or angiogenesis related molecules (e.g., mRNA moleculesor proteins). Preferably, the composition contains a pharmaceuticallyacceptable carrier. Suitable carriers include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The composition can be adapted for the mode ofadministration and can be in the form of, for example, a pill, tablet,capsule, spray, powder, or liquid.

The invention also features a variety of databases. These databasesinclude information on the effect of a compound on the expression oractivity of cancer or angiogenesis related mRNA molecules or proteins.These databases may also be used in the development of combinationtherapies and in the selection of a preferred therapy for a particularpatient or class of patients.

In one such aspect, the invention features an electronic databaseincluding at least 5, 10, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹records of mRNA molecules or proteins (e.g., cancer or angiogenesisrelated molecules) correlated to records of compounds (e.g., signaltransduction inhibitors) and their ability to modulate the expression oractivity of mRNA molecules or proteins. Preferably, the databaseincludes records for at least 5, 10, 10², 10³, 10⁴, 10⁵, or 10⁶compounds. In yet other embodiments, the database includes records forat least one protein expressed by an open reading frame for at least0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% of the openreading frames in the genome of a mammal, such as a human.

In another aspect, the invention features a computer including adatabase of the invention and a user interface (i) capable of displayingone or more compounds (e.g., signal transduction inhibitors) thatmodulate the activity of an mRNA or protein whose record is stored inthe computer or (ii) capable of displaying one or more mRNA molecules orproteins whose expression or activity is modulated by a compound whoserecord is stored in the computer. The internal components of thecomputer typically include a processor coupled to a memory. The externalcomponents usually include a mass-storage device, e.g., a hard diskdrive; user input devices, e.g., a keyboard and a mouse; a display,e.g., a monitor; and optionally, a network link capable of connectingthe computer system to other computers to allow sharing of data andprocessing tasks. Programs may be loaded into the memory of this systemduring operation.

In various embodiments of any of the aspects of the invention, theanalysis of an expression profile includes comparing the expressionlevel of an mRNA or protein in the sample to the corresponding level ina control sample (e.g. a sample from a healthy patient). In otherembodiments, the expression level of an mRNA or protein in a sample fromcancerous or diseased cells in the mammal is compared to thecorresponding level in a sample from healthy cells in the mammal. In yetother embodiments, the expression profile includes the expression levelof pro-angiogenic mRNA molecules or proteins, such as vascularendothelial growth factor, transforming growth factor alpha,angiopoietin-1, plasminogen activator inhibitor-1, or anti-angiogenicmRNA molecules or proteins, such as thrombospondin-1. Other exemplaryangiogenesis related molecules are listed in FIGS. 1G and 3. Preferably,the expression profiling is performed using a DNA chip.

In certain embodiments, the expression profiling also includes thedetection of the presence or absence of polymorphic or mutant forms ofmRNA molecules or proteins. In preferred embodiments, the expression oractivity of an mRNA or protein that has a mutation associated withcancer or an angiogenesis related disease and that has a higher thannormal, normal, or even lower than normal level of expression isinhibited by a therapy of the invention. For subjects that express botha mutant form (e.g., a cancer-related form) and a wild-type form (e.g.,a form not associated with cancer) of an mRNA or protein, the therapypreferably inhibits the expression or activity of the mutant form by atleast 2, 5, 10, or 20-fold more than it inhibits the expression oractivity of the wild-type form.

In preferred embodiments, at least 1, 2, 3, 5, 10, or all of thecompounds in the combination therapy each modulate the expression oractivity of at least 2, 3, 5, 10, 15, 20, 30, or more mRNA molecules orproteins. In other preferred embodiments, the therapy modulates theexpression or activity of at least 2, 3, 5, 10, 15, 20, 30, or more mRNAmolecules or proteins. In some embodiments, a compound or therapyincreases the expression or activity of at least 2, 3, 5, 10, 15, 20,30, or more anti-angiogenic or cancer suppressor mRNA molecules orproteins and decreases the expression or activity of at least 2, 3, 5,10, 15, 20, 30, or more pro-angiogenic or oncogenic mRNA molecules orproteins. Preferably, the therapy modulates the expression or activityof at least 2, 3, 5, 10, 15, 20, 30, or more mRNA molecules or proteinsby at least 20, 40, 50, 75, 90, 100, 200, 500, or even 1000%. In otherpreferred embodiments, at least 20, 40, 50, 75, 90, 95, or 100% of themRNA molecules or proteins whose expression or activity is modulated bythe therapy are molecules that had altered expression levels compared tothe corresponding levels in healthy patients. Preferably, the therapymodulates the expression or activity of at least 20, 40, 50, 75, 90, 95,or 100% of all of the mRNA molecules or proteins that had alteredexpression levels compared to the corresponding levels in healthypatients. In another preferred embodiment, the therapy modulates theexpression or activity of at least 20, 40, 50, 75, 90, 95, or 100% ofall of the cancer or angiogenesis related mRNA molecules or proteinsthat had altered expression levels compared to the corresponding levelsin healthy patients.

In particular embodiments, the therapy includes at least 3, 5, 7, 10,15, 20 or more compounds, such as signal transduction inhibitors. Inparticular embodiments, the pharmaceutically active compounds in thetherapy only include signal transduction inhibitors. In otherembodiments, the therapy includes both signal transduction inhibitorsand other chemotherapeutic agents, such as cytotoxic agents orimmunotherapy agents. Preferred therapies include the signaltransduction inhibitor Herceptin or any other compound (e.g., amonoclonal or polyclonal antibody) that binds or inhibits Her2 (U.S.Pat. No. 6,165,464). Another preferred signal transduction inhibitor is^(Pr)Gleevec™ (imatinib mesylate, Novartis Pharmaceuticals Canada Inc.,Dorval, Quebec). Preferred combination therapies include Herceptinand/or ^(Pr)Gleevec™. Other exemplary therapies include an anti-vascularagent, cyclophosphamide, and/or vinblastine. It is also contemplatedthat the therapy can include an agent (e.g., naked DNA, a DNA vector, ora viral vector) that inactivates a gene that promotes cancer (e.g., anoncogene) or excessive angiogenesis or a gene that is expressed at ahigher than normal level in a subject, or the therapy can include anagent (e.g., naked DNA, a DNA vector, or a viral vector encoding an mRNAor protein of interest or containing a promoter that integrates upstreamof an endogenous gene of interest) that increases the expression of agene that inhibits cancer (e.g., a tumor suppressor gene) or excessiveangiogenesis or a gene that is expressed at a lower than normal level ina subject.

In preferred embodiments, the therapy is administered using an extendedrelease device. In certain embodiments, the therapy is administeredorally, intramuscularly, intravenously, subcutaneously, or byinhalation.

Exemplary cancers that can be treated, stabilized, or prevented usingthe above methods include prostate cancers, breast cancers, ovariancancers, pancreatic cancers, gastric cancers, bladder cancers, salivarygland carcinomas, gastrointestinal cancers, lung cancers, colon cancers,melanomas, brain tumors, leukemias, lymphomas, and carcinomas. Thecancer may or may not be a hormone related or dependent cancer (e.g., anestrogen or androgen related cancer). Benign tumors may also be treatedor prevented using the methods and compositions of the presentinvention. Preferably, the therapy inhibits angiogenesis of the cancer(e.g., inhibits the rate of blood cell formation or decreases the numberor size of blood vessels) by at least 10, 25, 40, 50, 60, 70, 80, 90,95, or 100%. Other exemplary angiogenesis related diseases that can betreated or prevented using the methods of the invention are listed inFIG. 4. Preferred mammals include humans and mammals of veterinaryinterest.

As used herein, by “higher than normal expression level” is meantexpression of an mRNA or protein at a level that is higher that theaverage expression level of the corresponding molecule in healthysubjects. In various embodiments, the expression level is at least 20,40, 50, 75, 90, 100, 200, 500, or even 1000% higher than the level incontrol subjects.

By “lower than normal expression level” is meant expression of an mRNAor protein at a level that is lower that the average expression level ofthe corresponding molecule in healthy subjects. In various embodiments,the expression level is at least 20, 40, 50, 75, 90, 95, or 100% lowerthan the level in control subjects. In some embodiments, the expressionof the mRNA or protein is not detectable.

By “modulate expression or activity” is meant to either increase ordecrease expression or activity, for example, of a protein or nucleicacid sequence, relative to control conditions. The modulation inexpression or activity is preferably an increase or decrease of at least20, 40, 50, 75, 90, 100, 200, 500, or even 1000%. In variousembodiments, transcription, translation, mRNA or protein stability, orthe binding of the mRNA or protein to other molecules in vivo ismodulated by the therapy. The level of mRNA may be determined bystandard Northern blot analysis, and the level of protein may bedetermined by standard Western blot analysis, such as the analysesdescribed herein or those described by, for example, Ausubel et al.(Current Protocols in Molecular Biology, John Wiley & Sons, New York,2000). In one embodiment, the level of a protein is determined bymeasuring the level of enzymatic activity, using standard methods. Inanother preferred embodiment, the level of mRNA, protein, or enzymaticactivity is equal to or less than 20, 10, 5, or 2-fold above thecorresponding level in control cells that do not express a functionalform of the protein, such as cells homozygous for a nonsense mutation.In yet another preferred embodiment, the level of mRNA, protein, orenzymatic activity is equal to or less than 20, 10, 5, or 2-fold abovethe corresponding basal level in healthy cells that have not beenexposed to conditions that induce abnormal cell proliferation or thatinhibit apoptosis.

By a “dosage sufficient to modulate mRNA or protein expression oractivity” is meant an amount of a therapy that increases or decreasesmRNA or protein expression or activity when administered to a subject.Preferably, for a compound that decreases expression or activity, themodulation is a decrease in expression or activity that is at least 10%,30%, 40%, 50%, 75%, or 90% lower in a treated subject than in the samesubject prior to the administration of the inhibitor or than in anuntreated, control subject. In addition, preferably, for a compound thatincreases expression or activity, the amount of expression or activityof the mRNA or protein is at least 1.5-, 2-, 3-, 5-, 10-, or 20-foldgreater in a treated subject than in the same subject prior to theadministration of the modulator or than in an untreated, controlsubject.

By “treating, stabilizing, or preventing cancer” is meant causing areduction in the size of a tumor, slowing or preventing an increase inthe size of a tumor, increasing the disease-free survival time betweenthe disappearance of a tumor and its reappearance, preventing an initialor subsequent occurrence of a tumor, or reducing an adverse symptomassociated with a tumor. In one preferred embodiment, the percent ofcancerous cells surviving the treatment is at least 20, 40, 60, 80, or100% lower than the initial number of cancerous cells, as measured usingany standard assay. Preferably, the decrease in the number of cancerouscells induced by administration of a therapy of the invention is atleast 2, 5, 10, 20, or 50-fold greater than the decrease in the numberof non-cancerous cells. In yet another preferred embodiment, the numberof cancerous cells present after administration of a therapy is at least2, 5, 10, 20, or 50-fold lower than the number of cancerous cellspresent after administration of a vehicle control. Preferably, themethods of the present invention result in a decrease of 20, 40, 60, 80,or 100% in the size of a tumor as determined using standard methods.Preferably, at least 20, 40, 60, 80, 90, or 95% of the treated subjectshave a complete remission in which all evidence of the cancerdisappears. Preferably, the cancer does not reappear or reappears afterat least 5, 10, 15, or 20 years. In another preferred embodiment, thelength of time a patient survives after being diagnosed with cancer andtreated with a therapy of the invention is at least 20, 40, 60, 80, 100,200, or even 500% greater than (i) the average amount of time anuntreated patient survives or (ii) the average amount of time a patienttreated with another therapy survives.

By an “estrogen-related cancer” is meant a cancer that is modulated byestrogen. Examples of estrogen-related cancers include, withoutlimitation, breast cancer and ovarian cancer. Her2 is overexpressed inmany estrogen-related cancers (U.S. Pat. No. 6,165,464).

By an “androgen-related cancer” is meant a cancer that is modulated byandrogen. An example of androgen-related cancers is prostate cancer.

By “cancer related gene” is meant a gene associated with an altered riskfor a cancer or an altered prognosis for a cancer. Exemplary cancerrelated genes that promote cancer include oncogenes; genes that enhancecell proliferation, invasion, or metastasis; genes that inhibitapoptosis; and pro-angiogenesis genes. Cancer related genes that inhibitcancer include, but are not limited to, tumor suppressor genes; genesthat inhibit cell proliferation, invasion, or metastasis; genes thatpromote apoptosis; and anti-angiogenesis genes.

By “angiogenesis” is meant the formation of new blood vessels and/or theincrease in the volume, diameter, length, or permeability of existingblood vessels, such as blood vessels in a tumor or between a tumor andsurrounding tissue. Angiogenesis is associated with a variety ofneoplastic and non-neoplastic disorders.

By “an angiogenesis related disease” is meant a disease associated withexcessive or insufficient blood vessel growth, an abnormal blood vesselnetwork, and/or abnormal blood vessel remodeling. For example,insufficient vascular growth can lead to decreased levels of oxygen andnutrients, which are required for cell survival. Angiogenesis alsocontributes to tumor growth. Other exemplary angiogenesis relateddiseases are listed in FIG. 4.

By “treating, stabilizing, or preventing an angiogenesis relateddisease” is meant modulating the formation of new blood vessels and/ormodulating the volume, diameter, length, permeability, or number ofexisting blood vessels. In preferred embodiments, an initial orsubsequent occurrence of an angiogenesis related disorder is preventedor an adverse symptom associated with an angiogenesis related disorderis reduced. Preferably, the methods of the present invention result in amodulation of 20, 40, 60, 80, 100, 500, or even 1000% in the volume,diameter, length, permeability, and/or number of blood vessels asdetermined using standard methods. Preferably, at least 20, 40, 60, 80,90, or 95% of the treated subjects have a complete remission in whichall evidence of the disease disappears. In another preferred embodiment,the length of time a patient survives after being diagnosed with anangiogenesis related disease and treated with a therapy of the inventionis at least 20, 40, 60, 80, 100, 200, or even 500% greater than (i) theaverage amount of time an untreated patient survives or (ii) the averageamount of time a patient treated with another therapy survives.

By “combination therapy” is meant a combination of two or more compoundsthat each modulate the expression or activity of an mRNA or protein(e.g., cancer or angiogenesis related molecules). The compounds maydirectly or indirectly modulate the expression or activity of the mRNAor protein. For example, a compound may indirectly modulate theexpression or activity of an mRNA or protein of interest by modulatingthe expression or activity of a molecule (e.g., a nucleic acid, protein,signaling molecule, growth factor, cytokine, or chemokine) that directlyor indirectly affects the expression or activity of the mRNA or proteinof interest. In some embodiments, the compounds inhibit cell division orinduce apoptosis. These compounds in the therapy may include, forexample, unpurified or purified proteins, antibodies, synthetic organicmolecules, naturally-occurring organic molecules, nucleic acidmolecules, and components thereof. The compounds in a combinationtherapy may be administered simultaneously or sequentially. Preferredcompounds are signal transduction inhibitors.

By “signal transduction inhibitor” is meant a compound that inhibits apathway involved in modulating intracellular events in response to anextracellular signal, such as the binding of a hormone to a cell-surfacereceptor. In some cases, the binding of a ligand to a cell-surfacereceptor modulates the phosphorylation state of the intracellular domainof the receptor or the phosphorylation state of proteins that interactwith the intracellular domain of the receptor. The change inphosphorylation state of the protein or other downstream proteins oftenresults in either the activation of a transcription factor and anincrease in gene expression or the inhibition of a transcription factorand a decrease in gene expression. Thus, a signal transduction inhibitortypically effects a change in the expression of one or more genes. Forexample, signal transduction inhibitors may inhibit an activity of oneor more molecules in a signal transduction pathway (e.g., anextracellular ligand such as a hormone, a cell-surface receptor, akinase, a phosphatase, or a transcription factor). In contrast, manycytotoxic agents nonspecifically kill proliferating cells and maynonspecifically inhibit the expression of a variety of genes.

By “purified” is meant separated from other components that naturallyaccompany it. Typically, a factor is substantially pure when it is atleast 50%, by weight, free from proteins, antibodies, andnaturally-occurring organic molecules with which it is naturallyassociated. Preferably, the factor is at least 75%, more preferably, atleast 90%, and most preferably, at least 99%, by weight, pure. Asubstantially pure factor may be obtained by chemical synthesis,separation of the factor from natural sources, or production of thefactor in a recombinant host cell that does not naturally produce thefactor. Proteins and small molecules may be purified by one skilled inthe art using standard techniques such as those described by Ausubel etal. (Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, 2000). The factor is preferably at least 2, 5, or 10 times as pureas the starting material, as measured using polyacrylamide gelelectrophoresis, column chromatography, optical density, HPLC analysis,or western analysis (Ausubel et al., supra). Preferred methods ofpurification include immunoprecipitation, column chromatography such asimmunoaffinity chromatography, magnetic bead immunoaffinitypurification, and panning with a plate-bound antibody.

By “mutation” is meant an alteration in a naturally-occurring orreference nucleic acid sequence, such as an insertion, deletion,frameshift mutation, silent mutation, nonsense mutation, or missensemutation. Preferably, the amino acid sequence encoded by the nucleicacid sequence has at least one amino acid alteration from anaturally-occurring sequence.

The present invention provides a number of advantages related to thetreatment and prevention of cancers and other angiogenesis relateddiseases. For example, the methods can be generally applied to thetreatment of malignant or benign tumors of any cell, tissue, or organtype. The simultaneous or sequential use of multiple therapeutic agents(e.g., signal transduction inhibitors) should greatly reduce theincidence of cancer and reduce the number of treated cancers that becomeresistant to therapy. In addition, therapeutic agents that are used aspart of a combination therapy may require a lower dose to treat a canceror angiogenesis related disease than the corresponding dose requiredwhen the therapeutic agents are used individually. The low dose of eachcompound in the combination therapy reduces the severity of potentialadverse side-effects from the compounds. Clinical trials involvingcombination therapies are also less expensive than performing multipleclinical trials that each involve only one chemotherapeutic agent.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J illustrate the effects of Herceptin on tumor growth,angiogenesis, and gene expression. FIG. 1A is a set of pictures of thearchitecture of the MDA-MB-361HK tumor vasculature on treatment day 15visualized by fluorescence microscopy. The scale bar indicates 100 μm.FIG. 1B is a graph illustrating that the mean vessel diameter wassignificantly reduced in the Herceptin-treated group (open circle, n=6)compared to the control antibody-treated group (filled circle, n=6).FIG. 1C is a bar graph showing that vascular permeability wassignificantly reduced in the Herceptin-treated group (open column, n=6)compared to the control antibody-treated group (filled column, n=6) atday 15. FIG. 1D is a graph illustrating that tumor size wassignificantly reduced in the Herceptin-treated group (open circle, n=6)compared to the control antibody-treated group (filled circle, n=6).FIG. 1E is a graph showing that survival was significantly increased inthe Herceptin-treated group (open column, n=6) compared to the controlantibody-treated group (filled column, n=6). FIG. 1F is a set of bargraphs showing that when the tumor reached 6 mm size, mean vesseldiameter, vessel volume, and vascular permeability were significantlyreduced in the Herceptin treated group (open column, n=6) compared tothe control antibody treated group (filled column, n=6), while vessellength was not significantly different. In FIG. 1B-1F, the data areshown as the mean±standard deviation, * P<0.05, Mann-Whitney U test(StatView, Abacus, Berkeley, Calif.), compared with the correspondingvalues in the Herceptin-treated group. In FIGS. 1B and 1D, data up totreatment day 15 are plotted since the first animal was sacrificed ontreatment day 15. FIG. 1G is a set of pictures of the expressionprofiling of 23 angiogenesis related genes performed using the HumanCancer/angiogenesis-2 GEArray kit (SuperArray Inc., Bethesda, Md.)according to manufacturer's instructions. Expression of vascularendothelial growth factor (VEGF), transforming growth factor alpha(TGF-α), angiopoietin-1 (Ang-1), and plasminogen activator inhibitor-1(PAI-1) decreased, and expression of thrombospondin-1 (TSP-1) increasedin the Herceptin treated tumors compared to the control tumors in vivo(left 2 columns). The expression profiles were confirmed by Northernblot analysis in vivo and in vitro (right 2 columns). “C” denotes thecontrol antibody-treated group, and “H” denotes the Herceptin-treatedgroup. Quantitative analysis was performed by densitometry. Theabundance of mRNA was normalized to that of β-Actin. FIG. 1H is apicture of the detection of HER2 expression using the A0485 antibody(Dako). HER2 was observed in the majority of MDA-MB-361-HK cells grownin the SCID mouse cranial window, primarily in the cell membrane in bothgroups. FIG. 1I is a picture of VEGF expression that was detected usingthe 3E7 antibody (gift from Drs. Rolf Brekken and Philip Thorpe). VEGFwas observed in the host stromal cells (arrows) as well as in the tumorcells in both groups. The images are from Herceptin treated tumors. Thescale bar indicates 50 μm. FIG. 1J is a table illustrating the effect ofHerceptin on tumor blood vessels and expression of angiogenic factors.

FIGS. 2A-2G are tables listing various angiogenesis inhibitors that maybe used in the therapies of the invention (Carmeliet and Jain, Nature407:249-257, 2000).

FIG. 3 is a table listing many proteins that activate or inhibitangiogenesis. The expression or activity of these factors can bemodulated using the methods of the invention (Carmeliet and Jain, Nature407:249-257, 2000).

FIG. 4 is a table listing many angiogenesis related diseases that can betreated or prevented using the methods and compositions of the invention(Carmeliet and Jain, Nature 407:249-257, 2000).

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that thesignal transduction inhibitor Herceptin modulates the expression ofmultiple cancer and angiogenesis related genes. Thus, Herceptin offersan attractive alternative to using multiple therapeutic agents tomodulate expression of these genes. Because Herceptin is a monoclonalantibody against human epidermal growth factor receptor 2 (HER2) whichis expressed in a variety of tumors, Herceptin may be generally used totreat numerous cancers and angiogenesis related diseases (Slamon et al.,N. Engl. J. Med. 344:783-792, 2001).

Based on this surprising discovery that Herceptin modulates theexpression of multiple pro- and anti-angiogenic factors, we expect othercompounds (e.g., other signal transduction inhibitors) to also modulatethe expression of multiple cancer and angiogenesis related genes. Thus,combination therapies that include two or more such compounds shouldhave increased drug efficacy because of the ability to modulate theexpression or activity of numerous cancer and angiogenesis relatedgenes. Preferred signal transduction inhibitors or preferred combinationtherapies (e.g., combinations of signal transduction inhibitors) can beselected for a particular patient by determining the expression profileof cancer or angiogenesis related genes in the patient and selecting atherapy that (i) inhibits the expression of genes that promote cancer orangiogenesis and that are expressed at a high level in the patientand/or (ii) promotes the expression of genes that inhibit cancer orangiogenesis and that are expressed at a low level in the patient.Preferably, the signal transduction inhibitor or combination therapynormalizes the expression or activity of the majority or all of thecancer or angiogenesis related genes that are expressed at an alteredlevel in the patient and has negligible effect on other genes.

These methods are described further below.

Inhibition of Angiogenesis by Herceptin

The ability of Herceptin treatment to induce normalization andregression of the vasculature of a HER2 over-expressing human breastcarcinoma was tested in vivo. For better in vivo growth of tumors forthis analysis, the MDA-MB-361HK cell line (Genentech Inc., SanFrancisco, Calif.) was derived from MDA-MB-361, a HER2 over-expressinghuman breast cancer cell line derived from a brain metastasis.MDA-MB-361HK tumor xenografts were established by injection of asingle-cell suspension into the dorsal flank of female SCID micesupplemented with an estrogen pellet (17-β-estradiol 0.36 mg, InnovativeResearch, Sarasota, Fla.) inserted subcutaneously a few days before cellinjection. Tumors were serially transplanted up to four generations.Cranial windows were prepared in 8-10 week old female SCID mice aspreviously described (Jain et al., in Tumor Models In Cancer Research,ed. Teicher, Humana Press Inc., Totowa, N.J., pp. 647-671, 2001). Thesemice were bred and maintained in our gnotobiotic animal facility. A fewdays after estrogen supplementation, a piece of MDA-MB-361HK tumor,approximately 1 mm in diameter, was implanted into the cranial windows.Approximately 14 days after tumor implantation, when the tumor becamewell vascularized (approximately 2 mm in diameter; referred to astreatment day 0), we began treating the animals with intraperitonealinjection of either Herceptin or control human IgG (Genentech), 30mg/kg, every 3 days. Treatment continued until the tumor diameterreached approximately 6 mm, the size of the window. Then, the mouse wassacrificed, and the tumor was resected for histological and molecularanalyses. Animal survival was derived from the time interval between theinitiation of treatment and animal sacrifice. Tumor size, vesseldensity, mean vessel diameter, and vessel volume of the cranial windowtumor were analyzed every 3 days from treatment day 0 using intravitalfluorescence microscopy and quantified as previously described (Jain etal., supra). For visualization of blood vessels, 10 mg/ml fluoresceinisothiocyanate labelled dextran solution (M_(r), 2,000,000; SigmaChemical Co., St. Louis, Mo.) was injected via a tail vein cannula.Vascular permeability to albumin was measured on treatment day 0, day15, and the day the tumor reached 6 mm, as previously described (Jain etal., supra). Mice were injected with a bolus (100 μl) of 1%tetramethylrhodamine labelled bovine serum albumin (Molecular Probes,Eugene, Oreg.) in saline via the tail vein.

Based on the above analysis, Herceptin treatment significantly reducedthe diameter and volume of tumor vessels compared to control antibodytreatment, while not significantly affecting the length of the vessels(FIGS. 1A and 1B). Vascular permeability was also significantly reducedby Herceptin treatment (FIGS. 1C and 1J; 8.8±5.7 and 1.6±1.1×10⁻⁷ cm s⁻¹at day 15 in the control and herceptin groups, respectively). Thus, thevessels in the Herceptin treated tumors more closely resembled a normalphenotype. Tumor growth was delayed and animal survival wassignificantly extended (FIGS. 1D, 1E, and 1J). These vascular effectswere not tumor-size dependent as evidenced by the sustained differencesat maximum (6 mm) tumor size (FIG. 1F). The more efficient blood vesselnetwork resulting from Herceptin treatment more closely resembles normalnetworks, and thus, may improve drug delivery to previously inaccessibleregions.

Modulation of Multiple Pro- and Anti-Angiogenic Factors by Herceptin

Based on the finding that the vascular effect of Herceptin was verystable throughout the course of the study, together with the knowndiversity of human epidermal growth factor receptor pathways (Yarden etal., Nat. Rev. Mol. Cell Biol. 2:127-137, 2001), we hypothesized thatHerceptin treatment affects multiple pro- and anti-angiogenic factors.To test this hypothesis, we examined 23 angiogenesis related genes usingthe Human Cancer/angiogenesis-2 GEArray kit (SuperArray Inc., Bethesda,Md.) according to manufacturer's instructions. For this analysis, thegene expression of tumors in vivo and in vitro was measured in thepresence and absence of Herceptin. In particular, tumors were removedfrom Herceptin and control antibody treated mice once the tumors reached6 mm in size. The tumor tissue was then homogenized, and the cells werelysed. To collect mRNA, the lysate was applied to a spin columncontaining an oligo-T matrix to bind the polyA termini of the mRNA. ThemRNA was then eluted from the matrix, and the mRNA was reversedtranscribed in the presence of [³²P]-dCTP to generate radiolabelledcDNA. A 1 μg sample of cDNA was hybridized to the chip overnight, andthen the radioactive cDNA bound to the chip was detected. To study theeffect of Herceptin on tumor cells in vitro, MDA-MB-361HK cells wereplated at a density of 2×10⁶ cells/10 cm dish overnight. The cells weretreated with 50 μg/ml Herceptin or control human IgG for 72 hours, andthe cell lysate was collected for Northern blot analysis and for DNAchip analysis, as described above.

This expression profiling demonstrated that VEGF, transforming growthfactor alpha (TGF-α), angiopoietin-1 (Ang-1), and plasminogen activatorinhibitor-1 (PAI-1) was decreased in Herceptin treated tumors. Incontrast, expression of thrombospondin-1 (TSP-1) increased in Herceptintreated tumors compared to control tumors in vivo (FIGS. 1G and 1J).These results were subsequently confirmed by Northern blot analysis(FIG. 1G).

HER2 signaling is known to control VEGF and PAI-1 expression. HER2 mayaffect TGF-α level through interactions with HER1, and may possiblymediate TSP-1 expression through pathways similar to Ras. To ourknowledge, no correlation has been reported between HER2 and Ang-1. Thevascular effects of Herceptin in the present study are consistent withthe reports that Ang-1 increases vessel diameter (Thurston et al.,Science 286:2511-2514, 1999), whereas TSP-1 decreases vessel diameter(Bornstein, J. Clin. Invest. 107:929-934, 2001). Vascular permeabilityis possibly affected by TSP-1 through recruitment of perivascular cells,balanced by the decrease in Ang-1. In this study, Northern blot analysisindicated that VEGF expression was decreased by Herceptin treatment invitro but not in vivo, suggesting VEGF compensation from host cells.Indeed, VEGF expression was observed by immunohistochemistry in hoststromal cells as well as in the tumor cells (FIGS. 1H and 1I). Also,cultured tumor cells expressed lower levels of Ang-1 and PAI-1 andexpressed a higher level of TSP-1 in vitro compared to in vivo tumortissues. The Herceptin mediated change in PAI-1 expression was greaterin vivo compared to in vitro. We speculate that Herceptin treatment mayhave affected PAI-1 from the host through modulation of tumor derivedTGF-α. These results collectively indicate significant contribution oftumor-host interaction in the expression of pro- and anti-angiogenicfactors.

Selection of Therapies

Instead of, or in addition to, Herceptin, other signal transductioninhibitors can be used for the treatment or prevention of cancers andangiogenesis related diseases. For example, additional therapeuticagents can be administered to inhibit other angiogenic factors, such asangiogenic factors from host cells. Thus, individual or multiple signaltransduction inhibitors can be used to achieve stable, long-term vesselnormalization and regression.

For the selection of a preferred signal transduction inhibitor or apreferred combination therapy for a particular patient, the effect ofother compounds (e.g., known signal transduction inhibitors such asthose listed in FIGS. 2A-2G or compounds other than signal transductioninhibitors) on gene expression may be determined. For example, theability of other compounds to modulate the expression of angiogenesisrelated genes or other cancer related genes may be determined using aDNA chip, as described above for Herceptin. In one exemplary approach, atumor cultured in vitro or a tumor in an animal model (e.g., a SCIDmouse) can be contacted with the compound. Exemplary doses for theadministration of an anti-cancer or angiogenesis compound to a SCIDmouse include the following: 50 mg/kg (morning) and 100 mg/kg (evening)p.o. for ^(Pr)Gleevec™, 1 mg/mouse i.p. every 2 days for C225, and 20mg/kg/day p.o for ZD 1839. Then, cells from the tumor are lysed, andmRNA is collected from the lysate and reverse transcribed to generateradiolabelled cDNA. The cDNA is applied to the HumanCancer/angiogenesis-2 GEArray kit or to any other DNA chip. For example,cDNA arrays that contain other known cancer or angiogenesis relatedmolecules (e.g., oncogenes or tumor suppressors) or that contain aportion or all of the cDNA molecules expressed by a particular mammal(e.g. a human) can be prepared using standard methods (Marrack et al.,Current Opinion in Immunology 12, 206-209, 2000; Harkin, Oncologist.5:501-507, 2000; Pelizzari et al., Nucleic Acids Res. 28(22):4577-4581,2000). Because DNA chips are commercially available for a variety ofcell types, the radiolabelled cDNA can also be applied to a DNA chipthat contains cDNA molecules from the same cell or tissue type as thetumor. The hybridization of the tumor-derived cDNA molecules to the DNAchip is detected using standard methods. Alternatively, the ability of acompound to modulate the expression of mRNA molecules or proteins can bedetected using standard Northern or Western analysis.

The lists of mRNA molecules or proteins that are expressed at alteredlevels in the presence of various compounds can be stored in a database.This database can be used to select individual or combination therapiesfor the treatment of a particular patient. For example, the expressionprofiling analysis described above is performed on cells from a patientwho may be at risk for cancer or an angiogenesis related disease orperformed on cancerous or diseased cells from a patient with cancer oran angiogenesis related disease. The expression of cancer orangiogenesis related genes in the patient sample is compared to thecorresponding expression level in a control sample. If desired, thepatient sample may also be analyzed for the presence of mRNA or proteinmolecules that contain a mutation associated with cancer or alteredangiogenesis (i) using DNA chip or Northern analysis with hybridizationprobes specific for the mutant or wild-type forms or (ii) using anantibody specific for the mutant or wild-type forms.

Next, one or more compounds are selected from the database that (i)inhibit the expression or activity of mRNA molecules or proteins thatpromote cancer or undesired angiogenesis that are expressed at a higherthan normal level in the patient sample or (ii) promote the expressionor activity of mRNA molecules or proteins that inhibit cancer orundesired angiogenesis that are expressed at a lower than normal levelin the patient sample. Additionally, compounds may be selected thatinhibit mRNA molecules or proteins that have a mutation that promotescancer or an angiogenesis related disease and that have an altered ornormal level of expression. The database can be used to select theindividual or combination therapy that (i) modulates the greatest numberof mRNA molecules or proteins that have altered expression levels in thepatient or that have mutations associated with cancer or alteredangiogenesis and/or (ii) modulates the least number of mRNA molecules orproteins that have normal expression levels in the patient. The selectedindividual or combination therapy should have high drug efficacy andproduce few, if any, adverse side-effects.

As an alternative to the patient-specific analysis described above, DNAchips can be used to compare the expression of mRNA molecules in aparticular type of early or late-stage tumor (e.g., breast cancer cells)or angiogenesis related disease tissue to the expression in normaltissue. Based on this analysis, an individual or combination therapy forpatients with this type of tumor or disease can be selected to modulatethe expression of the mRNA or proteins that have altered expression inthis tumor or diseased tissue.

In addition to being used to select a therapy for a particular patientor group of patients, expression profiling can be used to monitor thechanges in mRNA and/or protein expression that occur during treatment.For example, expression profiling can be used to determine whether theexpression of cancer or angiogenesis related genes has returned tonormal levels. If not, the dose of one or more compounds in the therapycan be altered to either increase or decrease the effect of the therapyon the expression levels of the corresponding cancer or angiogenesisrelated gene(s). In addition, this analysis can be used to determinewhether a therapy affects the expression of other genes (e.g., genesthat are associated with adverse side-effects). If desired, the dose orcomposition of the therapy can be altered to prevent or reduce undesiredside-effects.

Exemplary Angiogenesis Related Molecules that can be Modulated Using theMethods of the Invention

In addition to the angiogenesis related molecules mentioned above, thereare numerous other factors that promote or inhibit angiogenesis and thatcan be modulated using the therapies of the invention (FIG. 3). Forexample, many growth factors and cytokines exert chemotactic, mitogenic,modulatory, or inhibitory activities on endothelial cells, smooth musclecell, and fibroblasts and, therefore, can be expected to participate inangiogenic processes. The process involves the concerted action ofproteolytic enzymes, extracellular matrix components, cell adhesionmolecules, and vasoactive factors. Factors modulating growth,chemotactic behavior, and/or functional activities of smooth musclecells include Activin A, Adrenomedullin, aFGF, ANF, Angiogenin,Angiotensin-2, Betacellulin, bFGF, CLAF, ECDGF (endothelial cell-derivedgrowth factor), ET (Endothelins), Factor X, Factor Xa, HB-EGF, Heartderived inhibitor of vascular cell proliferation, IFN-gamma, IL1, LDGF(Leiomyoma-derived growth factor), MCP-1, MDGF (macrophage-derivedgrowth factor, monocyte-derived growth factor), nitric oxide (NO),Oncostatin M, PD-ECGF, PDGF, Prolactin, prostacyclin, Protein S, SDGF(smooth muscle cell-derived growth factor), SDMF (Smooth musclecell-derived migration factor), Tachykinins, TGF-beta, andThrombospondin.

Factors modulating growth, chemotactic behavior, and/or functionalactivities of vascular endothelial cells include aFGF, ANF, Angiogenin,Angiostatin, Angiotropin, AtT20-ECGF, B61, bFGF, bFGF inducing activity,CAM-RF, ChDI, CLAF, Cox-2, ECG, ECI, EDM, EGF, EMAP, Neurothelin,Endostatin, Endothelial cell growth inhibitor, Endothelialcell-viability maintaining factor, Ephrins, Epo, FGF-5, IGF-2, HBNF,HGF, HUAF, IFN-gamma, IL1, K-FGF, LIF, MD-ECI, MECIF, MMPs, NO,Oncostatin M, PAI-2, PD-ECGF, PDGF, PF4, PlGF, Prolactin, TIMPs,TNF-alpha, TNF-beta, Transferrin, urokinase, and VEGI. Proteins thathave angiogenesis activity in vivo include fibroblast growth factors,Angiogenin, Angiopoietin-1, EGF, HGF, VEGA, VEGB, VEGC, VEGD, VEGE,VEGF, TNF-alpha, TGF-beta, PD-ECGF, PDGF, IGF, IL8, and Growth hormone.Fibrin fragment E also has angiogenic activity. In addition,Angiopoietin-1 plays a prominent role in vasculogenic and angiogenicprocesses. PF4 and a 16 kDa fragment of Prolactin are inhibitory invivo.

Angiogenic activities also include a number of other compounds such asprostaglandins E1 and E2, steroids, heparin, 1-butyryl glycerol(monobutyrin) secreted by adipocytes, and many undefined derivatives ofthe arachidonic acid metabolism. The biologically active principleextracted from some carcinoma cells and identified as nicotinic amide isalso a potent angiogenic compound in several bioassays.

Signal Transduction Inhibitors

In addition to Herceptin, there are numerous other signal transductioninhibitors that can be used in the methods of the present invention toprevent or treat cancer or angiogenesis related diseases. Examples ofother signal transduction inhibitors include ^(Pr)Gleevec™ (also calledST1571), IMC-C225 (from ImClone), ZD 1839 (from AstraZeneca), IGF-1, andplatelet-factor 4. Other exemplary signal transduction inhibitorytherapies include anti-estrogen therapies such as an adreno-ovariectomysurgical procedure and compounds that inhibit estrogen activity (e.g.,tamoxifen or an LHRH analog). Still other signal transduction inhibitorytherapies include anti-androgen therapies such as surgical castrationand compounds that inhibit androgen activity (e.g., ˜50 mg dailybicalutamide, cyproterone acetate, dexamethasone, or an LHRH analog).

Anti-angiogenesis tyrosine kinase inhibitors can also be used tomodulate tumor cells that have receptors for these kinases. For example,SU6668 can be used to inhibit PDGF, FGF, and VGEF. SU5416 and ZD4190inhibit VGEF. Additionally, IMC-IC11 (DC101, from ImClone) is ananti-VGEF antibody that inhibits VGEF. Other exemplary angiogenesisinhibitors are listed in FIGS. 2A-2G.

Other Compounds for Inclusion in Individual or Combination Therapies

In general, additional drugs for the treatment of cancer or angiogenesisrelated diseases may be identified from large libraries of both naturalproduct or synthetic (or semi-synthetic) extracts or chemical librariesaccording to methods known in the art. Those skilled in the field ordrug discovery and development will understand that the precise sourceof test extracts or compounds is not critical to the methods of theinvention.

Accordingly, virtually any number of chemical extracts or compounds canbe screened for their effect on the activity or expression of cancer orangiogenesis related factors. Examples of such extracts or compoundsinclude, but are not limited to, plant-, fungal-, prokaryotic- oranimal-based extracts, fermentation broths, and synthetic compounds, aswell as modification of existing compounds. Numerous methods are alsoavailable for generating random or directed synthesis (e.g.,semi-synthesis or total synthesis) of any number of chemical compounds,including, but not limited to, saccharide-, lipid-, peptide-, andnucleic acid-based compounds. Synthetic compound libraries arecommercially available from Brandon Associates (Merrimack, N.H.) andAldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant, and animal extractsare commercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). Inaddition, natural and synthetically produced libraries are produced, ifdesired, according to methods known in the art, e.g., by standardextraction and fractionation methods. Furthermore, if desired, anylibrary or compound is readily modified using standard chemical,physical, or biochemical methods.

When a crude extract is found to modulate the activity or expression ofa cancer or angiogenesis related factor, further fractionation of thepositive lead extract is necessary to isolate chemical constituentresponsible for the observed effect. Thus, the goal of the extraction,fractionation, and purification process is the careful characterizationand identification of a chemical entity within the crude extract.Methods of fractionation and purification of such heterogeneous extractsare known in the art. If desired, compounds shown to be useful agentsfor the treatment of cancer or an angiogenesis related disease arechemically modified according to methods known in the art. Compoundsidentified as being of therapeutic value are subsequently analyzed usingany standard animal model of angiogenesis or cancer known in the art.

Assays and Animal Models for the Testing of Therapies of the Invention

If desired, the therapies of the invention can be tested for theireffect on cancer and angiogenesis using the SCID mouse model describedherein. Additionally, there are numerous standard assays and animalmodels that can be used to determine the efficacy of particulartherapies for inhibiting angiogenesis [Auerbach et al, Dev. Biology 41:391-394 (1974); Brown et al, Laboratory Investigation 75(4): 539-555(1996); Castellot et al., Journal of Cellular Physiology 127: 323-329(1986); Gaudric et al, Research 24: 181-188 (1992); Greenblatt andShubik, Journal of the National Cancer Institute 41: 111-124 (1968);Hayek et al., Microvasc. Research 41: 203-209 (1991); Lichtenberg et al,Pharmacol Toxicology 84(1): 34-40 (1999); Lichtenberg et al, PharmacolToxicology 81(6): 280-284 (1997); Mourad et al., British Journal ofDermatology 123: 21-28 (1990); Nissanov et al, Laboratory Investigation73(5): 734-739 (1995); Okamura et al, Biochemical and BiophysicalResearch Communications 186: 1471-1479 (1992); Olivo et al., Anat. Rec.234: 105-115 (1992); O'Reilly et at, Cell 79(2): 315-328 (1994);O'Reilly et al, Cell 88: 277-285 (1997); Passaniti et al., Lab. Invest.67: 519-528 (1992); Peek et al, Experimental Pathology 34: 35-40 (1988);Polyerini et al, Methods in Enzymology 198; 440-450 (1991); Ribatti etal., Journal of Vascular Research 34(6): 455-463 (1997); Sato et al,Journal of Investigative Dermatology 95: 85S-89S (1990); Wilting J etal, Anat. Embryology 183: 259-271 (1991)]. The individual andcombination therapies can also be tested in standard human clinicaltrials.

Administration of Therapies

A therapy of the invention may be administered to humans, domestic pets,livestock, or other animals with a pharmaceutically acceptable diluent,carrier, or excipient, in unit dosage form.

The compounds optionally may be administered as pharmaceuticallyacceptable salts, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like.

The chemical compounds for use in such therapies may be produced andisolated as described herein or by any standard technique known to thosein the field of medicinal chemistry. Conventional pharmaceuticalpractice may be employed to provide suitable formulations orcompositions to administer the identified compound to patients sufferingfrom cancer or an angiogenesis related disease or at increased forcancer or an angiogenesis related disease. Administration may beginbefore or after the patient is symptomatic.

Any appropriate route of administration may be employed. For example,the therapy may be administered either directly to the tumor (forexample, by injection) or systemically (for example, by any conventionaladministration technique). Preferably, the therapy is administered usinga controlled-release microchip, microparticle extended-releaseformulation, polymeric nanoparticle, or transdermal delivery system (asdescribed, for example, in LaVan et al., Nature Reviews 1:77-84, 2000 orSantini et al., Nature 397:335-338, 1999). Administration of thecompounds may also be oral, topical parenteral, intravenous,intraarterial, subcutaneous, intramuscular, intracranial, intraorbital,ophthalmalic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, or intranasal. Alternatively, thecompounds may be administered as part of a suppository. Therapeuticformulations may be in the form of liquid solutions or suspensions; fororal administration, formulations may be in the form of tablets orcapsules; and for intranasal formulations, in the form of powders, nasaldrops, or aerosols. The compounds in a combination therapy may beadministered simultaneously or sequentially. For example, one or morecompounds in a combination therapy can be administered until thecompound(s) normalize the blood vessel network of the tumor and therebyincrease the accessibility of the tumor to other therapeutic agents, andthen one or more additional compounds can be administered instead of, orin addition to, the originally administered compound(s). The dosage ofthe therapeutic compounds in a pharmaceutically acceptable formulationdepends on a number of factors, including the size and health of theindividual patient. The dosage to deliver may be determined by oneskilled in the art. For example, compounds that are administered as partof a combination therapy of the invention are typically administered ata dose equal to or at least 25, 50, or 75% lower than the correspondingdose for the compound when it is used individually. An exemplary dosingregimen for Herceptin includes a 4 mg/kg loading dose followed by aweekly dose of 2 mg/kg. Other suggested doses include 200-400 or 400-600mg/body/day for ^(Pr)Gleevec™, a 400 mg/m² loading dose followed by aweekly dose of 250 mg/m² for IMC-C225, and 150-1000 mg/body/day forZD1839.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” ((19thed.) ed. A. R. Gennaro AR., 1995, Mack Publishing Company, Easton, Pa.).Formulations for parenteral administration may, for example, containexcipients, sterile water, saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

If desired, treatment with a compound identified according to themethods described above, may be combined with more traditional therapiesfor cancer or an angiogenesis related disease (e.g., cytotoxic agents,radiation therapy, or surgical removal of cancerous cells).

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications, patent applications, and patents mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

1. A method for selecting a combination therapy for the treatment,stabilization, or prevention of a cancer or an angiogenesis relateddisease in a mammal, said method comprising the steps of: (a) analyzingthe expression profile of more than one mRNA and/or protein in a sampleobtained from said mammal; and (b) selecting a therapy that comprisestwo or more compounds that each (i) decrease the expression level oractivity of an mRNA or protein that has a higher than normal expressionlevel in said mammal and/or (ii) increase the expression level oractivity of an mRNA or protein that has a lower than normal expressionlevel in said mammal.
 2. A method for preventing, delaying, or treatinga cancer or an angiogenesis related disease in a mammal, said methodcomprising the steps of: (a) analyzing the expression profile of morethan one mRNA and/or protein in a sample obtained from said mammal; (b)selecting a therapy that comprises two or more compounds that each (i)decrease the expression level or activity of an mRNA or protein that hasa higher than normal expression level in said mammal and/or (ii)increase the expression level or activity of an mRNA or protein that hasa lower than normal expression level in said mammal; and (c)administering said therapy to said mammal in an amount sufficient totreat, stabilize, or prevent said cancer or angiogenesis relateddisease.
 3. A method for stratification of subjects involved in aclinical trial of a combination therapy comprising two or more compoundsfor the treatment, stabilization, or prevention of a cancer or anangiogenesis related disease in a mammal, said method comprising thesteps of: (a) analyzing the expression profile of more than one mRNAand/or protein in a sample obtained from a subject; and (b) determiningthe presence of a lower or higher than normal expression level for morethan one mRNA and/or protein in said sample before, during, or aftersaid clinical trial, wherein the presence of said expression profile insaid subject places said subject into a subgroup for said clinicaltrial.
 4. The method of claim 1, wherein said therapy comprises a signaltransduction inhibitor.
 5. The method of claim 4, wherein all of thepharmaceutically active compounds in said therapy are signaltransduction inhibitors.
 6. The method of claim 4, wherein said therapycomprises a compound other than a signal transduction inhibitor.
 7. Amethod for selecting a signal transduction inhibitor for the treatment,stabilization, or prevention of a cancer or an angiogenesis relateddisease in a mammal, said method comprising the steps of: (a) analyzingthe expression profile of one or more mRNA molecules and/or proteins ina sample obtained from said mammal, wherein the expression or activityof said mRNA molecule or protein can be modulated by a signaltransduction inhibitor; and (b) selecting a signal transductioninhibitor that (i) decreases the expression level or activity of an mRNAor protein that has a higher than normal expression level in said mammaland/or (ii) increases the expression level or activity of an mRNA orprotein that has a lower than normal expression level in said mammal. 8.A method for preventing, delaying, or treating a cancer or anangiogenesis related disease in a mammal, said method comprising thesteps of: (a) analyzing the expression profile of one or more mRNAmolecules and/or proteins in a sample obtained from said mammal, whereinthe expression or activity of said mRNA molecule or protein can bemodulated by a signal transduction inhibitor; (b) selecting a signaltransduction inhibitor that (i) decreases the expression level oractivity of an mRNA or protein that has a higher than normal expressionlevel in said mammal and/or (ii) increases the expression level oractivity of an mRNA or protein that has a lower than normal expressionlevel in said mammal; and (c) administering said signal transductioninhibitor to said mammal in an amount sufficient to treat, stabilize, orprevent said cancer or angiogenesis related disease.
 9. A method forstratification of subjects involved in a clinical trial of a signaltransduction inhibitor for the treatment, stabilization, or preventionof a cancer or an angiogenesis related disease in a mammal, said methodcomprising the steps of: (a) analyzing the expression profile of one ormore mRNA molecules and/or proteins in a sample obtained from a subject,wherein the expression or activity of said mRNA molecule or protein canbe modulated by a signal transduction inhibitor; and (b) determining thepresence of a lower or higher than normal expression level for one ormore mRNA molecules and/or proteins in said sample before, during, orafter said clinical trial, wherein the presence of said expressionprofile in said subject places said subject into a subgroup for saidclinical trial.
 10. The method of claim 7 or 8, further comprisingselecting another signal transduction inhibitor that (i) decreases theexpression level or activity of another mRNA or protein that has ahigher than normal expression level in said mammal and/or (ii) increasesthe expression level or activity of another mRNA or protein that has alower than normal expression level in said mammal.
 11. The method ofclaim 7 or 8, further comprising repeating step (b) until signaltransduction inhibitors are selected for the modulation of theexpression or activity of all the cancer or angiogenesis related mRNAmolecules or proteins that have lower or higher than normal expressionlevels in said mammal.
 12. The method of claim 4 or 7, wherein saidsignal transduction inhibitor is Herceptin.
 13. The method of claim 4 or7, wherein said signal transduction inhibitor is ^(Pr)Gleevec™.
 14. Themethod of claim 1 wherein said therapy comprises five compounds.
 15. Themethod of claim 1 or 7, wherein step (a) comprises comparing theexpression level of an mRNA or protein in said sample to thecorresponding level in a control sample.
 16. (canceled)
 17. The methodof claim 1 or 7, wherein at least one of said compounds or said signaltransduction inhibitors modulates the expression of at least 3 mRNAmolecules or proteins.
 18. (canceled)
 19. The method of claim 1 or 7,wherein the expression profile comprises the expression level of mRNAmolecules or proteins selected from the group consisting of vascularendothelial growth factor, transforming growth factor alpha,angiopoietin-1, plasminogen activator inhibitor-1, and thrombospondin-1.20. (canceled)
 21. (canceled)
 22. (canceled)
 23. The method of claim 1or 7, wherein said therapy inhibits angiogenesis of said cancer by atleast 25%.
 24. (canceled)
 25. The method of claim 1 or 7, wherein saidmammal or said subject is a human.
 26. (canceled)
 27. (canceled)